Field of the Invention
The present invention relates to the field of infectious disease, in particular to therapeutics for the treatment of Clostridium difficile infection.
Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual compositions or methods used in the present invention may be described in greater detail in the publications and patents discussed below, which may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance or the prior art effect of the patents or publications described.
Clostridium difficile infection (CDI) is worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural microbial gut flora and allow the Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of the disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic resistant bacteria has created an urgent need for new therapies1. Here we describe targeting the cysteine protease domain (CPD) of the C. difficile major virulence factor toxin B (TcdB) that is critical for the pathophysiological function of the toxin within host cells. Using an activity-based probe for the toxin CPD2, we performed a high-throughput fluorescence polarization screen and identified a number of potent inhibitors, including one bioactive compound, ebselen, which is currently in use in human clinical trials for other indications. This drug showed activity in biochemical and cell-based studies. Most importantly, treatment of a mouse model of CDI that closely resembles the human infection3 confirmed a significant therapeutic benefit in the form of reduced disease pathology in host tissues. Our results confirm that a non-antibiotic drug that blocks function of a major bacterial virulence factor can modulate the histopathology of disease and therefore should immediately be explored as a potential therapeutic for the treatment of CDI.
CDI is the leading cause of nosocomial diarrhea and the sole cause of pseudomembrane colitis4. With a yearly average of over a quarter million hospitalizations in the US5, this infectious disease places a burden of over $1 billion on the US health care system6. Furthermore, the mortality rate in patients with CDI is high: 6.9% at 30 days after diagnosis and 16.7% at 1 year7. Skyrocketing numbers of cases in the past decade (139,000 in 2002 versus 336,600 in 2012), increasing recurrence, and a rise in the number of strains resistant to almost all available antibiotics is creating a significant public health threat8. Only a handful of options are available to treat CDI infection, antibiotics being the main clinical practice. Recurrence rates for CDI are as high as 25%, with most patients returning to the clinic for antibiotic treatment as soon as diarrhea recurs9,10. By disrupting the growth cycle of bacteria, antibiotics rapidly select for resistant subpopulations11. As such, the rates of nosocomial antibiotic-resistant opportunistic pathogens causing infections have more than doubled in the past decade12,13. Although fecal bacteriotherapy has proven to be a highly effective treatment for CDI patients14, it remains controversial from the aspect of drug regulation with the long-term effects on human health remaining uncharacterized15.
An important potential strategy to combat bacterial pathogens is to block the ability of bacteria to harm the host by inhibiting bacterial virulence factors16. This strategy would help limit antibiotic use and in turn decrease the rate of emergence of resistant strains. In contrast to antibiotic treatment, targeting virulence factors may help to promote regrowth of the commensal gut flora, a key factor in mediating resistance to CDI17. The pathology of CDI is mediated exclusively by large clostridial toxins (TcdA and B) and only toxigenic C. difficile causes disease18,19. All disease-causing strains, including the epidemic BI/NAP1/02720, carry the gene for toxin B21. The toxin is comprised of a putative receptor binding domain, a transmembrane domain, a CPD, and a glucosyltransferase domain (GTD)22. When endocytosed, and upon acidification within vesicles, it undergoes membrane translocation, exposing the CPD to the mammalian-specific cytosolic sugar, 1D-myo-inositol hexakisphosphate (IP6)23. The allosteric binding of IP6 activates the CPD to autocatalytically cleave the GTD domain which induces toxicity by irreversible glucosylation of the Rho-Rac family of small GTPases in host intestinal epithelial cells. This event results in rearrangement of the actin cytoskeleton, acute inflammation, massive fluid secretion, and finally necrosis of the mucosal layer24, 25 causing symptoms such as severe diarrhea, fever, nausea, abdominal pain/tenderness, and loss of appetite26. Severe cases of CDI are characterized by pseudomembranous colitis; perforations of the colon; and sepsis27, and in acute situations, the mortality rate can be as high of 40%. Even though the pathology of CDI is mediated by toxins, none of the currently available treatments target these major virulence factors.
Specific Patents and Publications
Puri et al., “Rational design of inhibitors and activity-based probes targeting Clostridium difficile virulence factor TcdB,” Chem Biol. 2010 Nov. 24; 17(11):1201-11, discloses the rational design of covalent small molecule inhibitors of TcdB CPD and the identification of compounds that inactivate TcdB holotoxin function in cells. Such peptides do not contain an alpha-beta unsaturated structure.
Zhao et al., “A Novel Antioxidant Mechanism of Ebselen Involving Ebselen Diselenide, a Substrate of Mammalian Thioredoxin and Thioredoxin Reductase,” Oct. 18, 2002 The Journal of Biological Chemistry, 277, 39456-39462, discloses the following reaction and dimer compound:

The paper discloses that the above reaction occurs when ebselen reacts with its selenol forming an ebselen diselenide. This compound is, as indicated in the title of the paper, is a substrate of mammalian thioredoxin and thioredoxin reductase; the authors propose a novel mechanism for ebselen antioxidant activity. This antioxidant activity is disclosed as important to ebselen's neuroprotective effects.